Sc 6420

Reagents were purchased from Sigma-Aldrich unless specified otherwise. Antibodies used in this study were as follow: anti-APP 6E10 (SIG-39320, Covance), anti-Aβ42 12F4 (SIG-39142, Covance), mouse anti-Tau Tau12 (SIG-39416, Covance), mouse anti-Tau HT7 (MN1000, Thermo Scientific Pierce), mouse anti-phosphorylated Tau AT8 (MN1020, Thermo Scientific Pierce), rabbit anti-Tau H150 (SC-5587, Santa Cruz Biotechnology), rabbit anti-Tau (A0024, Dako), rabbit anti-clusterin H330 (SC-8354, Santa Cruz Biotechnology), goat anti-clusterin M18 (SC-6420, Santa Cruz Biotechnology), goat anti-clusterin C18 (SC-6419, Santa Cruz Biotechnology), mouse anti-BIN1 99D (05-449, Millipore), rabbit anti-Gapdh (G9545), rabbit anti-actin (A2066), and mouse anti-Flag (F1804), mouse anti-myc 9E10 (SC-40, Santa Cruz Biotechnology), mouse and goat IgG control (SC-2015 and SC-2028, Santa Cruz Biotechnology), donkey anti-mouse IgG (H+L) IRDye 800 (926-32212, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 680 (926-68073, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 800 (926-32213, LI-COR Biosciences), and donkey anti-goat IgG (H+L) IRDye 680 (926-68074, LI-COR Biosciences).

Proteins from equal volume cell culture media samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were probed with primary antibodies overnight at 4 °C with gentle shaking. Membranes were then incubated for 1 h with secondary antibody–horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:10,000. Specific signals were developed for 1 min using enhanced chemiluminescence (enhanced chemiluminescence (ECL) kit components 1 and 2, GE Healthcare UK Limited, Buckinghamshire, UK). Chemiluminescence was visualized by exposure of photographic film (LAS-4000, Fujifilm, Tokyo, Japan).
The following primary antibodies were used: a human CLU-specific antibody, which binds to the alpha-chain in the unreduced protein, (sc-6420, Santa Cruz Biotechnology, Santa Cruz, CA, USA); a strep-tag-specific antibody (IBA 2-1507-001, IBA Lifesciences, Göttingen, Germany); an his-tag-specific antibody (ab18184, Abcam, Cambridge, UK).